Hwang Grace H, Hopkins Jessica L, Jordan Philip W
Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health.
Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health;
J Vis Exp. 2018 Feb 26(132):56736. doi: 10.3791/56736.
Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.
染色质铺展技术已被广泛用于评估配子发生过程中各种蛋白质的动态定位，特别是在精子发生过程中。这些技术能够观察减数分裂事件（如同源染色体配对、联会和DNA修复）期间蛋白质和DNA的定位模式。虽然文献中已经描述了一些方案，但由于胎儿卵巢中减数分裂起始的时间，使用哺乳动物减数分裂前期卵母细胞的一般染色质铺展技术有限且困难。相比之下，可以从幼年雄性小鼠中收集减数分裂前期精母细胞，产量更高，无需显微切割。然而，由于幼年和成年睾丸中减数分裂和减数分裂后生殖细胞群体的异质性，很难获得特定阶段的纯同步细胞群体。对于减数分裂的后期阶段，评估处于减数分裂I（MI）或减数分裂II（MII）的卵母细胞是有利的，因为可以从成年雌性小鼠中收集成熟卵母细胞群体，并在培养中刺激其恢复减数分裂。在这里，描述了使用从胎儿、新生和成年卵巢中解剖的卵母细胞进行减数分裂染色质铺展制备的方法，并配有视频演示。哺乳动物卵母细胞中的染色体错分离事件很常见，尤其是在MI期间。这些技术可用于评估和表征不同突变或环境暴露在卵子发生各个阶段的影响。由于卵子发生和精子发生之间存在明显差异，本文所述技术对于增进我们对哺乳动物卵子发生以及减数分裂期间染色体和蛋白质动态的性别二态性特征的理解非常宝贵。
